Abstract 2909: Targeting Polθ suppresses CCNE1-amplified cancers via transcription-replication conflicts mediated DNA damage and RAD51 toxicity

Abstract Background: CCNE1 amplification is prevalent in various cancers, including ovarian, endometrial, and breast cancers, and is associated with elevated cyclin E levels, chemotherapy resistance, and poor prognosis. Despite its clinical relevance, no approved therapeutic strategies currently target CCNE1-amplified tumors. DNA polymerase θ (Polθ) has emerged as a promising therapeutic target in DNA repair-deficient cancers, such as HRD tumors. However, its function in oncogene-amplified cancers characterized by high replication stress and endogenous DNA damage remains underexplored. Methods & Results: Public dataset analyses revealed a positive correlation between Polθ and CCNE1 expression across various tumor types in both clinical samples and cancer cell lines. Elevated Polθ expression was linked to worse survival outcomes exclusively in CCNE1-high tumors. Using a doxycycline (Dox)-inducible CCNE1 overexpression HeLa cell model and a panel of CCNE1-amplified, CCNE1-gain, and CCNE1-low cell lines, we demonstrated that CCNE1 overexpression upregulated Polθ expression, enhanced MMEJ repair activity, and increased sensitivity to Polθ inhibitors. Polθ inhibition, achieved through chemical inhibitors or shRNA knockdown, significantly impaired cell viability and exacerbated DNA damage, replication stress, and genomic instability in CCNE1-overexpressing cells. RNA-seq analysis revealed that CCNE1 overexpression promoted replication and transcription initiation events, while immunofluorescence and proximity ligation assays confirmed transcription-replication conflicts (TRCs) as a key outcome of CCNE1 overexpression. Polθ inhibition exacerbated TRC-associated DNA damage, both at the end of early S phase and after a round of cell cycle. In CCNE1-high cells, Polθ inhibition resulted in the accumulation of large RAD51 foci. Unlike normal CCNE1-high cells, where RAD51 foci resolved during cell cycle progression, Polθ inhibition caused persistent foci, indicating failed homologous recombination (HR) repair. Colocalization studies implicated genome G4 sites in this process, suggesting that Polθ compensates for stalled RAD51-mediated repair at G4-prone regions. Conclusions: Polθ inhibition suppresses CCNE1-amplified tumors by amplifying replication stress, DNA damage, and genomic instability. We propose that Polθ supports tumor cell survival in CCNE1-amplified contexts by resolving TRC-associated DNA damage and toxic HR intermediates. Additionally, CCNE1-induced TRCs primarily occur at G4-prone regions, where Polθ mitigates RAD51 repair failure, underscoring its critical role in maintaining genomic stability under oncogene-induced replication stress. Citation Format: Chuancong Zhu, Yu Xu, Xuejiao Zhao, Gordon B. Mills, Ding Ma, Qinglei Gao, Yong Fang. Targeting Polθ suppresses CCNE1-amplified cancers via transcription-replication conflicts mediated DNA damage and RAD51 toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2909.